Cellulose is a major component of plant material. It is the basis for the structural integrity of plants and is often found in a lignocellulose matrix composed of cellulose, hemicelluloses, and lignin. Applications employing cellulose take advantage of either its structural properties (fibers, textiles, paper, etc.) or of its carbohydrate nature, producing D-glucose, cellobiose and/or cellulose oligomers.
Lignocelluloses are readily available from agriculture and forestry including byproduct streams from cereals, corn, sugar cane, sugar beet, timber, etc. Plants that are optimized for their lignocellulose content and yield (“energy crops”) will likely contribute as an important resource in the near future.
Cellulases comprise a structurally and functionally diverse class of glycohydrolases acting on cellulose. Cellulases are found in bacteria, archea, fungi and plants. Having in common the hydrolytic cleavage activity of glycosidic bonds present in cellulose polymers or oligomers, they differ in substrate specificity, mode of action, and enzyme parameters, including processivity, pH and temperature optima. Most cellulases act on β-1,4-linkages between two glucose moieties. However other linkages found in lignocelluloses may also be hydrolysed. Cellulases can be subdivided by their mode of action into endo- and exo-enzymes. Endoglucanases introduce random cleavages into the cellulose polymer, thereby reducing the degree of polymerization. Exo-enzymes, like cellobiohydrolases, work in a successive mode of action, releasing cellobiose (D-glucose-β-1,4-D-glucopyranoside) from the reducing or non-reducing end of the polymer.
The CAZY Database [Cantarel B L, Coutinho P M, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res 37:D233-238 PMID: 18838391] holds, amongst others, a collection of known glucohydrolases including cellulose degrading enzymes (i.e. cellulases). In this database enzymes are classified to different GH-classes according to structural elements. Several GH classes include endoglucanases, in particular the classes GH5, GH7, GH9, GH12, GH16, GH45, GH48, GH61 and GH74. Despite the high diversity within some of the GH classes, members of one GH class often have similar physical and enzymatic parameters. This allows general statements to be made like substrate specificity, pH range, stability, or catalytic efficiency for members of a certain GH class.
Cellulose-degrading microorganisms often produce and secrete a complex mixture of cellulases. For instance, in the secretome of Trichoderma reesei 7 endoglucanases have been identified belonging to 6 different GH classes (Cel5A, Cel7B, Cel12A, Cel45A, Cel61A, Cel61B, Cel74A). The different endoglucanases show a spectrum of properties (Karlsson J, Siika-aho M, Tenkanen M, Tjerneld F. Enzymatic properties of the low molecular mass endoglucanases Cel12A (EG III) and Cel45A (EG V) of Trichoderma reesei. J Biotechnol. 2002 Oct. 9; 99(1):63-78. PubMed PMID: 12; Karlsson J, Momcilovic D, Wittgren B, Schülein M, Tjerneld F, Brinkmalm G. Enzymatic degradation of carboxymethyl cellulose hydrolyzed by the endoglucanases Cel5A, Cel7B, and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45Acore from Trichoderma reesei. Biopolymers. 2002 January; 63(1):32-40. PubMed PMID: 11754346.). The two predominant endoglucanases, EGI (Cel7B, GH7) and EGII (Cel5A), are considered to be the most active enzymes thereof.
The synergistic activity of cellulolytic enzymes allows the efficient breakdown of complex substrates (B. Henrissat, H. Driguez, C. Viet & M. Schülein: Synergism of Cellulases from Trichoderma reesei in the Degradation of Cellulose; Nature Biotechnology 3, 722-726 (1985) doi:10.1038/nbt0885-722) and precludes the replacement of a component of one structural class by an enzyme from a second fold, when at the same time the hydrolytic efficiency needs to be kept at maximum level (Non-equivalency of different EGs). A simple replacement by another GH class enzyme is not always possible. Generally speaking, members of endoglucanases from the GH5 family (including EGs from thermophilic bacteria) show higher thermostability compared to endoglucanases of the GH7 family; nevertheless, the application of a thermostable GH7 family protein is often advantageous for high hydrolysis rates.
Many applications of endoglucanases were reported, as part of complex enzyme mixtures as single enzyme activities. Cellulases are important for making cellulose-derived biofuels. After cutting and, optionally, chemical and/or physical pretreatment, lignocelluloses are incubated with cellulases to release sugar monomers that are further processed. Process conditions need to be adapted to optimize hydrolysis rates, yields and/or stability. Higher temperatures are often preferred in these processes but require more thermostable enzymes. Simultaneous saccharification and hydrolysis (SSF) processes require cellulolytic enzymes that are active under fermentative conditions. Consolidated bioprocessing (CBP) further requires the combination of enzyme properties, in order to have enzyme production, saccharification and fermentation done in a single step.
Other applications of endoglucanases aim only on a partial hydrolysis or modification of cellulose fibers (fiber modification, biopolishing, biostoning, etc.). Endoglucanases used therefore need to work and/or be stable at elevated temperatures, extreme (e.g. alkaline, acid) pH, and chemical conditions (e.g. laundry, detergents, proteases, solvents, etc.). Fiber damage must be minimized for such applications. Endoglucanases can also assist in the separation of non-cellulosic fractions from the fiber material in pulping processes (pulp & paper production) or improve rheological properties of process streams. Detergent stability and protease resistance can be seen as a product of increased stability of the enzyme structure, a property that is also connected to increased thermal stability. Endoglucanases also find applications in food and feed processing (breweries, wine production, oil recovery from press cake, baking, dough preparation. Often sterilization or pasteurization requires higher temperatures. For shortening of processing times the operational stability of the endoglucanase can be advantageous.
Endoglucanase I proteins (Cel7B) derived from fungi of the genus Trichoderma (anamorph Hypocrea) show high degrees of identity and are considered mesophilic. The most stable members of endoglucanases from the GH family 7 reported are native enzymes from Humicola insulens (Cel7B) and Fusarium oxysporum (eg1) (U.S. Pat. No. 5,912,157). According to said report, EGI does not exhibit activity above 60° C. There is thus a need in the field for the provision of more thermostable endoglucanases from the GH family 7.
It was reported that some endoglucanases can be thermally inactivated at higher temperatures (Dominguez J M, Acebal C, Jimenez J, de la Mata I, Macarron R, Castillon M P. Mechanisms of thermoinactivation of endoglucanase I from Trichoderma reesei QM 9414. Biochem J. 1992 Oct. 15; 287 (Pt 2):583-8.). The authors of said study also attempted re-activation of thermoinactivated endoglucanase, but this required harsh conditions involving 8 M urea and further agents. Effects described as productive refolding were shown on other proteins than endoglucanases [Zhang N, Suen W C, Windsor W, Xiao L, Madison V, Zaks A. Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution. Protein Eng. 2003 August; 16(8):599-605.], but to the knowledge of the inventors not for endoglucanases, in particular endoglucanases of GH7. It is believed in the art that thermoinactivated endoglucanases are of little use in industrial breakdown of cellulose. On the other hand, elevated thermostability is often desired for endoglucanases, in particular for enzymes of fungal origin. So far, only some improvements for endoglucanases of GH12 and GH45 were reported. Thermostable endoglucanases have been reported from the structural folds of GH5 and GH48. Said endoglucanases substantially differ with respect to their kinetic properties and substrate preference from the endoglucanases of the GH7 class.
In summary, there is a need for processive endoglucanases, particularly of the GH7 family, with superior temperature profiles. It would furthermore be desirable to achieve good productivity from their expression host. The need is further supported by the fact that many processes of industrial relevance run under harsh conditions and at elevated temperatures. A problem to be solved by the present invention is the provision of improved endoglucanases, particularly of endoglucanases with improved thermal properties. Further problems addressed and solved by this invention will become apparent from the sections below.